The goals of this proposal are to examine cellular mechanisms which regulate Na transport across renal tissue. Understanding Na transport is essential for understanding how the body maintains stable, long term blood pressure. Despite previous descriptions of individual molecular elements which regulate Na channels, the complete signaling cascades which contain these elements have not been described. Therefore, this application proposes to investigates, using patch clamp techniques supplemented by direct biochemical measurements, the signaling cascades which regulate sodium channels in a cellular model of the mammalian kidney principal cell, the Xenopus distal nephron cell line (A6). The specific aims for the proposed grant period will investigate three signaling cascades. The aims are: (1) examine the regulation of sodium channels by heterotrimeric G protein signaling cascades. Activation of an apical G protein inhibits Na channels through a mechanism which also involves protein kinase C, but little else is known about the pathway including which class of G protein is responsible for Na channel regulation; whether luminal receptors activate the G proteins; what molecules are activated by the G proteins; and what the final effects on Na channels are? (2) Investigate the mechanisms by which steroid hormones increase sodium channel activity. Methylation of excised, apical membranes induces sodium channel activity similar to the action of aldosterone and that the methylation reaction is augmented by G protein stimulation. Questions to be addressed for this aim are what is the target for methylation? Is it a small G protein like Ras? Is O- carboxy-methyl transferase activity altered by aldosterone? Is the activity of methyl transferase controlled by endogenous inhibitors like S-adenosyl homocysteine? (3) Investigate the mechanisms by which insulin-like growth factor 1 (IGF-1) Increase sodium channel activity. Determine the role of tyrosine kinases in the regulation of sodium channels. Both insulin and IGF-1 increase sodiu, transport and generalized inhibition of tyrosine kinases blocks the effect of IGF-1 and insulin. In addition, at least one subunit of the sodium channel is a target for IGF-1 mediated tyrosine phosphorylation. To further examine the role of insulin, IGF-1, and tyrosine kinases in regulating sodium channels, several questions will be answered is there any difference in the action of insulin and IGF-1 on A6 cells? Which Na channel subunits are tyrosine phosphorylated by IGF-1? Are there effects of tyrosine kinases in the absence of insulin or IGF-1?